The specific aims of the present proposal cover three major areas of the newly-identified phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the cerebral cortex. (I). Phosopholipid-sensitive Ca2+dependent protein kinase. The enzyme will be purified to homogeneity from the extract of pig (or bovine) cerebral cortex, with the use of several steps including chromatographies on DEAE-cellulose, controlledpore glass, phosphatidylserine-Affigel 102, dodecyl-Agarose (hydrophobic) and ATP-Agarose. Biochemical aspects of the enzyme, i.e. molecular and catalytic properties and regulatory mechanisms of Ca2+ and phospholipid/diolein, will be studied. Antibody against the enzyme will be produced, and the enzyme will be further characterized and differentiated from other protein kinases (activated by Ca2+/calmodulin, cyclic AMP or cyclic GMP) with the use of immunological and/or radioimmunochemical techniques. (II). Endogenous substrate proteins. Substrate proteins in the rat and/or guinea pig cerebral cortex for the enzyme will be further studied. They will be compared with endogenous proteins in the same tissue phosphorylated by other protein kinases. The major substrate proteins for the present phospholipid-sensitive Ca2+-dependent system will be purified to homogeneity. Antibodies against them will be raised, so that the immunological aspects of substrate proteins can be defined. (III). Effects of centrally active drugs. Pharmacological studies of the effects of CNS drugs, such as trifluoperazine and chlorpromazine, on phospholipid-sensitive Ca2+-dependent protein kinase and on its phosphorylation of endogenous proteins in the cerebral cortex will be carried out, and the results obtained will be compared with their effects on other protein phosphorylation systems. It is hoped that the present proposal can help establish this new Ca2+ effector system as (a) a major protein phosphorylation system (functioning independently of, or in a complementary manner with, other protein phosphorylation systems) closely involved in neuronal activities, and (b) a protential site of bioregulation and drug action.